PROJECT SUMMARY Both plants and animals rely on small pools of self-renewing stem cells to continually produce differentiated tissues. These stem cell populations act as a cellular reservoir that can be used to repair damage and generate new cell lineages. In plants, stem cells make up the apical meristems that continually produce organs, such as leaves, throughout their lifespans. The overarching goal of this proposal is to understand how a family of closely related transcription factors, the Class IIII HD-ZIP proteins, can pattern both the shoot apical meristem (SAM) and the leaves it gives rise to. Genetic studies have indicated that this family is not simply acting in a redundant fashion during this process, as certain combinations appear to uncover antagonistic roles as well. The study of how this family has diverged to play both redundant and antagonistic roles, as well as how together they specify and maintain stem cells and pattern differentiated organs are broad themes that are applicable to animal systems as well. This proposal seeks to combine genetic, genomic, imaging, and biochemical approaches to understand how the HD-ZIP III proteins acquired their unique and shared patterning roles in the Arabidopsis shoot. By combining these techniques, this study will provide a deep understanding of this gene family that will act as a model for studies on other families of closely related factors, which is a common theme in plant biology. Specifically, recombineering technologies coupled to confocal imaging will be used to characterize the tissue and cell type accumulation of each of the HD-ZIPIII proteins and assess if the differences in accumulation patterns affect their functions. A second aim will be to determine the genome wide differences in HD-ZIPIII target sites between the meristem and the developing leaves using a modified INTACT approach. Finally, specific protein interaction partners will be identified and their contributions to HD-ZIPIII diversity will be explored. Taken together these studies will provide one of the most in depth investigations into a closely related gene family in plants. The mechanisms and logic we find in our studies of the HD-ZIP III proteins can then be used as a framework to study similar gene families and processes in both plants and animals.